The goal of the proposed research is to uncover the molecular basis of channel opening and Ca2+ permeability of the skeletal muscle Ca2+ release channel (ryanodine receptor, RyR1). RyR1 is a 2,200 kDa ion channel that releases Ca2+ ions in response to an action potential from the sarcoplasmic reticulum (SR), an intracellular Ca2+-storing compartment in skeletal muscle. The release channel has a high conductance for monovalent (~800 pS with 250 mM K+ as conducting ion) and divalent cations (~150 pS with 50 mM Ca2+) yet is selective for Ca2+ (PCa/PK ~7). However, the molecular basis of the unique ion permeability properties is poorly understood. The principal hypothesis to be tested in the proposed research is that a combined experimental and computational approach will substantially increase our knowledge of the molecular determinants responsible for channel opening and the high ion transport rates of RyR1. Three specific aims are to (i) to apply a novel computational methodology of finding a protein conformational ensemble consistent with cryo-electron microscopy and sequence mapping data to build a structural model comprised of the 6 predicted transmembrane segments of RyR1, (ii) apply all-atom umbrella-sampling simulations to calculate the free energy profile of ion translocation through the pore of wild type, engineered and disease-associated RyR1 mutants, and (iii) develop multiscale modeling tools, which will involve the incorporation of atomistic details of ion-pore interactions into long time-scale discrete molecular dynamics, to directly quantify ionic currents in the channel. Pore mutants already available will provide the basis for modeling channel structure and the flow of ions. These include mutants linked to central core and multi minicore diseases. Computational data in turn will be critically tested by generating additional RyR1 mutants and determining their ion permeability properties in single channel recordings using the planar lipid bilayer method. As our studies progress we expect to gain new insights in the complex mechanism of RyR1 opening, ion conductance and selectivity and how these processes are altered by mutations in RyR1 linked to core myopathies.